In eukaryotic organisms, DNA methylation is catalyzed by an S-adenosyl-L-methionine (AdoMet)1-dependent DNA cytosine-C5 methyltransferase (DCMTase, EC 2.1.1.37). Methyl group transfer to the cytosine-C5 position occurs predominately within the cytosyl-guanosyl (CpG) context (Boyes, J., & Bird, A. P., 1991, DNA methylation inhibits transcription indirectly via a methyl-CpG binding protein, Cell 64:1123–1134). The genomic distribution of 5-methylcytosine (5-mC) dynamically changes throughout ontogeny (Razin, A., & Riggs, A. D., 1980, DNA methylation and gene function, Science 210:604–609; Kafri, T. et al., 1992, Developmental pattern of gene-specific DNA methylation in the mouse embryo and germ line, Genes and Dev. 6:705–714). The methylation state of a gene specifically affects transcription.
DCMTase is involved in mammalian development by way of an undefined process that can lead to gene regulation (reviewed in Jost, J. P., & Saluz, H. P., 1993, DNA Methylation: Molecular Biology and Biological Significance, Birkhauser Verlag, Basel). Proper DCMTase function is essential for viable development and for normal cellular activity (Li, E. et al., 1992, Targeted mutation of the DNA methyltransferase gene results in embyonic lethality, Cell 69:915–926).
Cytosine methylation is the predominant epigenetic event in the modification of eukaryotic DNA. To date only a single DCMTase has been identified in several metazoan organisms (Yoder, J. A., et al., 1996, New 5′ regions of the murine and human genes for DNA cytosine-5 methyltransferase, J. Biol. Chem. 271:31092–31097). The function most often identified with cytosine C5 methylation (5-mC) in higher eukaryotes is the regulation of transcription (Jost, J. P., & Saluz, H. P., 1993, DNA Methylation: Molecular Biology and Biological Significance, Birkhauser Verlag, Basel). Generally, hypermethylated genes are transcriptionally silent and inheritance of the proper genomic methylation pattern is critical to viable development as shown by DCMTase gene knock-outs in mice (Li, E., et al., 1992, Targeted mutation of the DNA methyltransferase gene results in embryonic lethality, Cell 69:15–926). Anti-sense directed inactivation of DCMTase mRNA as well as the incorporation of the cytosine analogs 5-azacytidine and 5-fluorocytidine into DNA interfere with DCMTase function and lead to cytological dysfunction (Ramachandani, S., et al., 1997, Inhibition of tumorigenesis by a cytosine-DNA, methyltransferase, antisense oligodeoxynucleotide, Proc. Natl. Acad. Sci. USA 94:684–689; Jones, P. A., 1985, Altering gene expression with 5-azacytidine, Cell 40:485–486).
Eukaryotic DCMTase cDNAs have been cloned and sequenced; five are from animal sources (mouse: Bestor, T., et al., 1988, Cloning and sequencing of a cDNA encoding DNA methyltransferase of mouse cells, J. Mol. Biol. 203:971–983; human: Yen, R. C., et al., 1992, Isolation and characterization of the cDNA encoding human DNA methyltransferase, Nucleic Acids Res. 20:2287–2291; chicken: Tajima, S., et al., 1995, Isolation and expression of a chicken DNA methyltransferase cDNA, J. Biochem. 117:1050–1057; frog: Kimura et al., 1996, Isolation and expression of a Xenopus laevis DNA methyltransferase cDNA, Journal of Biochemistry, 120:1182–1189; sea urchin: Aniello et al., 1996, Isolation of cDNA clones encoding DNA methyltransferase of sea urchin P. lividus: expression during embryonic development, Gene 178:57–61). These DCMTases are composed of a large amino-terminal domain and a smaller carboxy-terminal domain that contains many of the major motifs found in prokaryotic DCMTases (Posfai, J., et al., 1989, Predictive motifs derived from cytosine methyltransferases, Nucleic Acids Res 17:2421–2435). The amino-terminal domain has been implicated in nuclear localization to DNA replication foci during S-phase (Leonhardt, H., et al., 1992, A targeting sequence directs DNA methyltransferase to sites of DNA replication in mammalian nuclei, Cell 71:865–873), metal binding by zinc finger domains, and DNA binding (Bestor, T. H., 1992, Activation of the mammalian DNA methyltransferase by cleavage of a Zn binding regulatory domain, EMBO 11:2611–2617; Chuang, L. S., et al., 1996, Characterisation of independent DNA and multiple Zn-binding domains at the N terminus of human DNA-(cytosine-5) methyltransferase: modulating the property of a DNA-binding domain by contiguous Zn-binding motifs, Chia, J., and Li, B. F. L., J. Mol. Biol. 257:935–948).
Although the cellular processes that determine the genomic patterns of DNA methylation are not understood, DCMTase has an essential role in these processes. A basic understanding of the binding and catalytic DNA sequence specificity (discrimination) of the enzyme, and the factors which regulate this specificity are important. Since the mammalian enzyme is a relatively large, 183 kDa protein, DNA sequences flanking the cognate CpG may modulate the ability of the enzyme to methylate particular CpG sites. However, the CpG flanking sequence preferences of the enzyme, and its preference for single- and double-stranded substrates have not been rigorously addressed by previous investigators (Bestor, T. H. et al., 1992, CpG islands in mammalian gene promoters are inherently resistant to de novo methylation, GATA 9:48–53; Hepburn, P. A., et al., 1991, Enzymatic methylation of cytosine in DNA is prevented by adjacent O6-methylguanine residues, J. Biol. Chem. 266:7985–7987; Bolden, A. H., et al., 1986, Primary DNA sequence determines sites of maintenance and de novo methylation by mammalian DNA methyltransferases, Mol. Cell. Bio. 6:1135–1140; Pfeifer, G. P., et al., 1985, Mouse DNA-cytosine-5-methyltransferase: sequence specificity of the methylation reaction and electron microscopy of enzyme-DNA complexes, EMBO J. 4:2879–2884; Ward, C., et al., 1987, In vitro methylation of the 5′-flanking regions of the mouse b-globin gene, J. Biol. Chem. 262:11057–11063; Carotti, D., et al., 1986, Substrate preferences of the human placental DNA methyltransferase investigated with synthetic polydeoxynucleotides, Biochim. et Biophys. Acta. 866:135–143; Carotti D. et al., 1986, supra; Wang, R. Y. H., et al., 1984, Human placental DNA methyltransferase: DNA substrate and DNA binding specificity, Nucl. Acids Res. 12:3473–3490; Pfeifer et al., 1985, supra; Gruenbaum, Y., et al., 1982, Substrate and sequence specificity of a eukaryotic DNA methylase, Nature 295:620–622).
There is evidence that errors in the proper maintenance of genomic methylation are involved in aging and cancer. CpG islands are reported to become hypermethylated with age and may down-regulate expression of essential genes (Antequerra & Bird, 1993, Number of CpG islands and genes in human and mouse, Proceedings of the National Academy of Sciences, USA, 90:11995–11999; Nyce, J. W., 1997, Drug-induced DNA hypermethylation: A potential mediator of acquired drug resistance during cancer chemotherapy, Mutation Research 386:153–161) Amplification of DCMTase expression by an exogenous mammalian DCMTase gene induces tumorigenic transformation of NIH 3T3 mouse fibroblasts (Wu et al., 1993, Expression of an exogenous eukaryotic DNA methyltransferase gene induces transformation of NIH 3T3 cells, Proc. Natl. Acad. Sci., USA, 90:8891–8895). Human neoplastic cells and cells derived from different stages of colon cancer express up to 200-fold higher levels of DCMTase than normal (El-Deiry et al., 1991, High expression of the DNA methyltransferase-gene characterizes human neoplastic cells and progression stages of colon cancer, Proc. Natl. Acad. Sci., USA, 88:3470–3474). This contributes substantially to tumor development in a mouse model of intestinal neoplasia (Laird, P. W., et al., 1995, Suppression of intestinal neoplasia by DNA hypomethylation, Cell 81:197–205). Changes in DNA methylation and DCMTase activity appear early in oncogenesis (Belinsky, S. A., et al., 1996, Increased cytosine DNA-methyltransferase activity is target-cell-specific and an early event in lung cancer, Proc. Natl. Acad. Sci. USA 93:4045–4050).
Conversely, antisense oligonucleotides that interfere with expression of DCMTase may inhibit tumorigenesis (Ramachandani et al., 1997, Inhibition of tumorigenesis by a cytosine-DNA methyltransferase, antisense oligonucleotide, Proc. Natl. Acad. Sci., USA, 94:684–689; MacLeod & Szyf, 1995, Expression of antisense to DNA methyltransferase mRNA induces DNA demethylation and inhibits tumorigenesis, J. Biol. Chem. 270:8037–8043). The anticancer agent 5-aza-deoxycytidine functions by inhibiting the DCMTase (Jones, 1985, Altering gene expression with 5-azacytidine, Cell 40:485–486; Jutterman et al., 1994, Toxicity of 5-aza-2′-deoxycytidine to mammalian cells is mediated primarily by covalent trapping of DNA methyltransferase rather than DNA demethylation, Proc. Natl. Acad. Sci., USA, 91:11797–11801). Changes in DNA methylation and DCMTase activity early in oncogenesis (Belinsky, S. A., et al., 1996, supra) and the ability of DCMTase inhibitors to virtually abolish adenoma formation in mice (Laird, P. W., et al., 1995, supra) suggest that DCMTase inhibitors might be useful anticancer therapeutics (Szyf, M., 1996, The DNA methylation machinery as a target for anticancer therapy, Pharmacol. Ther. 70:1–37). 5-Aza-deoxycytidine is an irreversible, mechanism-based DCMTase inhibitor that has been used in patients with acute myeloid leukemia. Unfortunately, 5-Aza-deoxycytidine is unstable in solution and may be carcinogenic as well as mutagenic (Jones, P. A., 1996, DNA methylation errors and cancer, Cancer Res. 56:2463–2467). There is a need for DCMTase inhibitors that do not require incorporation into DNA and that are mechanistically unlike 5-aza-deoxycytidine (Belinsky, S. A., et al., 1996, supra; Szyf, M., 1996, supra; Jones, 1996, supra). A keen understanding of how DCMTase functions in vitro can be the basis for better strategies to both activate and inhibit the enzyme to correct developmental disorders like cancer.
Enzymes that catalyze one carbon additions to C5 of pyrimidines define a class of enzymes with similar chemistry (Ivanetich, K. M., & Santi, D. V., 1992, 5,6-Dihydropyrimidine adducts in the reactions and interactions of pyrimidines with proteins, Prog. Nucleic Acid Res. Mol. Biol. 42:127–156). The bacterial DNA cytosine C5 methyltransferase, M.HhaI (38 kDa Mr), modifies the internal cytosine in GCGC and has an ordered Bi Bi kinetic mechanism in which DNA binds first (Wu, J. C., & Santi, D. V., 1987, Kinetic and catalytic mechanism of HhaI methyltransferase, J. Biol. Chem. 262:4778–4786). Catalysis involves nucleophilic attack of an active site cysteine at the C6 position of the cytosine which, in the absence of the cofactor, leads to exchange of the C5 hydrogen. A M.HhaI-DNA cocrystal structure suggests that a catalytic intermediate exists that involves the translocation of the target cytosine to an extrahelical position covalently-bound to an active site cysteine (Klimasauskas, S., et al., 1994. HhaI methyltransferase flips its target base out of the DNA helix, Cell 76:357–369). Methyl transfer from AdoMet is followed by β-elimination to regenerate the active enzyme (Wu & Santi, 1987, supra; Osterman, D. G., et al., 1988, 5-Fluorocytosine in DNA is a mechanism-based inhibitor of HhaI methylase, Biochemistry 27:5204–5210).
A recent kinetic study of a highly homogeneous, unproteolyzed preparation of DCMTase from mouse erythroleukemia cells (MEL) further characterized the interactions of the enzyme with DNA and AdoMet (Flynn, J., et al., 1996, Murine DNA cytosine-C5 methyltransferase: Pre-steady- and steady-state kinetic analyses with regulatory DNA sequences, Biochemistry 35:7308–7315). The invention disclosed herein descriptively accounts for the previously reported complexities in kinetic behavior and identifies a potent single-stranded oligonucleotide inhibitor that binds to the enzyme at a distinct regulatory site.
There is a need for molecules which modulate the methylation of DNA for the reasons discussed above. In addition, molecules which inhibit DNA methylation can be useful for preventing drug resistance acquired by subjects undergoing cancer chemotherapy.
Drug-induced DNA hypermethylation is regarded as a potential mediator of this acquired drug resistance (Nyce, J. W., 1997, Drug-induced DNA hypermethylation: A potential mediator of acquired drug resistance during cancer chemotherapy, Mutation Research 386:153–161).